The t(8;21) is one of the most frequent chromosomal translocations associated with acute myeloid leukemia (AML). It fuses the DNA binding domain of AML1 (RUNX1) to nearly all of ETO (MTG8). However, an alternatively spliced form of AML1-ETO termed 9a, which deletes the C-terminal NH3 and NH4 conserved domains of ETO, is much more leukemogenic when expressed in murine stem and myeloid progenitor cells. The C-terminal domains of ETO/MTG family members also suffer point mutations in solid tumors and the NH4 domain of MTG16 is deleted by the inv(16)(p13.3q24.3), which fuses MTG16 to GLIS2. The short latency and high penetrance of AML caused by AE9a and the inactivation of NH4 in other cancers, suggests that proteins associating with the C-terminus of MTGs and AML1-ETO provide a barrier to tumor/leukemia development. Yeast two-hybrid (Y2H) screens using MTG family members identified factors that regulate RNA Pol II pausing and elongation that associated with the C-terminus of MTGs, which is deleted in the AE9a isoform. Analysis of Mtg16-/- and Mtg8-/- knockout mice suggested that these factors are key mediators of the knock out phenotypes and that MTGs are negative regulators of transcription elongation. In addition, cancer-associated point mutations disrupt the association between MTGs or AML1-ETO and elongation factors, further suggesting that the association of AML1-ETO or MTGs with elongation factors mediates the barrier to leukemia. Therefore, we hypothesize that AE9a releases elongation factors from negative regulation to promote AML development. This hypothesis will be directly tested genetically using cancer-associated point mutations that affect binding to elongation factors and by the use of innovative methods that measure RNA polymerase II pausing and elongation. We also hypothesize that these data begin to explain why t(8;21) containing cells are so sensitive to inhibitors that target transcriptional elongation and we will directly test this hypothesis by measuring how these drugs affect transcription in t(8;21)-containing cells.